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Inactivation of Gene α-1,3-Galactosyltransferase in Bovine Aortic Smooth Muscle Cells Using CRISPR-Cas9
Inactivation of Gene α-1,3-Galactosyltransferase in Bovine Aortic Smooth Muscle Cells Using CRISPR-Cas9
Tuesday, March 13, 2018
Grand Ballroom Foyer (CenturyLink Convention Center)
People who are bitten by Amblyomma americanum can develop a severely allergic reaction to red meat. The main reason that causes this kind of allergy is the presences of α-1,3-galactosyl epitopes on the membranes of most mammalian cells. Since human beings do not create galactose-alpha-1,3-galactose (alpha-gal), the patients’ immune system is free to start attacking alpha-gal whenever red meat is consumed. The epitope transferase coded geneα-1,3-galactosyltransferase (GGTA1) was already identified and can be knocked out to solve the red meat allergy. The GGTA1 gene has been successfully knocked out in pigs. However, there is no evidence to show the GGTA1 gene has been knocked out in beef cattle. In this study, we utilized bovine aortic smooth muscle cells (BAOSMC) as the module for the research of GGTA1 gene knockout.
The clustered regularly interspaced short palindromic repeats (CRISPR) systems are widely used in performing targeted genome editing in cultured cells. The use of CRISPR-associated (Cas) systems as an RNA-programmable DNA targeting and editing platform is followed by a synthetic single-guide RNA (sgRNA), which can simply edit genome sequence like other tools such as transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs).
We used the gRNA design tool and selected 5'-GGCCTGACGGTTTTCGCCGT-3' as the target gRNA sequence from the coding DNA sequence of Bos taurus alpha-galactosyltransferase 1 (glycoprotein). The gRNA was constructed in the pSpCas9 BB-2A-GFP (PX458) vector provided by GenScript USA Inc. Vectors were amplified and transfected into BAOSMC by GenePORTER2 transfection reagent when the cells were 80% confluency. Green fluorescent can be viewed after 24 hours transfection. The transfection efficiency can reach about 70% to 80%.
Cells were collected in PBS at a pH of 7.4 after 24 hours transfection. Total protein was extracted then the enzyme-linked immunosorbent assay was used to examine the GGTA1 production. By normalized with the total protein concentration, the GGTA1 protein level in the transfected cells was 17.9% ± 7.25% lower (P< 0.05) than in the control cells, showing a significant inhibition of GGTA1 gene expression in the cells by CRISPR-Cas9 gene edition method.
Our preliminary data shows that the gRNA sequence chosen was suitable for the GGTA1 gene knockout in BAOSMC. Moreover, the CRISPR-Cas9 system was proven it can be applied in the genome editing of bovine cells.
The clustered regularly interspaced short palindromic repeats (CRISPR) systems are widely used in performing targeted genome editing in cultured cells. The use of CRISPR-associated (Cas) systems as an RNA-programmable DNA targeting and editing platform is followed by a synthetic single-guide RNA (sgRNA), which can simply edit genome sequence like other tools such as transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs).
We used the gRNA design tool and selected 5'-GGCCTGACGGTTTTCGCCGT-3' as the target gRNA sequence from the coding DNA sequence of Bos taurus alpha-galactosyltransferase 1 (glycoprotein). The gRNA was constructed in the pSpCas9 BB-2A-GFP (PX458) vector provided by GenScript USA Inc. Vectors were amplified and transfected into BAOSMC by GenePORTER2 transfection reagent when the cells were 80% confluency. Green fluorescent can be viewed after 24 hours transfection. The transfection efficiency can reach about 70% to 80%.
Cells were collected in PBS at a pH of 7.4 after 24 hours transfection. Total protein was extracted then the enzyme-linked immunosorbent assay was used to examine the GGTA1 production. By normalized with the total protein concentration, the GGTA1 protein level in the transfected cells was 17.9% ± 7.25% lower (P< 0.05) than in the control cells, showing a significant inhibition of GGTA1 gene expression in the cells by CRISPR-Cas9 gene edition method.
Our preliminary data shows that the gRNA sequence chosen was suitable for the GGTA1 gene knockout in BAOSMC. Moreover, the CRISPR-Cas9 system was proven it can be applied in the genome editing of bovine cells.